This invention relates generally to methods and compositions useful in treating intervertebral disc impairment in humans and other mammals. More particularly, this invention concerns compositions useful in restoring hydrodynamic function and stimulating cell proliferation and extracellular matrix production in intervertebral discs that have been compromised by injury, degenerative disease, congenital abnormalities, and/or the aging process.
Compositions of the invention may be injectable, and may include growth factors, bioactive agents, and living cells. The compositions are useful for restoring, improving, or augmenting hydrodynamic function of the intervertebral disc, increasing intervertebral disc height, and stimulating cell proliferation and/or extracellular matrix production in intervertebral discs.
The human vertebral column (spine) comprises a plurality of articulating bony elements (vertebrae) separated by soft tissue intervertebral discs. The intervertebral discs are flexible joints which provide for flexion, extension, and rotation of the vertebrae relative to one another, thus contributing to the stability and mobility of the spine within the axial skeleton.
The intervertebral disc is comprised of a central, inner portion of soft, amorphous mucoid material, the nucleus pulposus, which is peripherally surrounded by an annular ring of layers of tough, fibrous material known as the annulus fibrosus. The nucleus pulposus and the annulus fibrosus together are bounded on their upper and lower ends (i.e., cranially and caudally) by vertebral end plates located at the lower and upper ends of adjacent vertebrae. These end plates, which are composed of a thin layer of hyaline cartilage, are directly connected at their peripheries to the lamellae of the inner portions of the annulus fibrosus. The lamellae of the outer portions of the annulus fibrosus connect directly to the bone at the outer edges of the adjacent vertebrae.
The soft, mucoid nucleus pulposus contains chondrocytes, which produce fibrils of collagen (primarily Type II collagen, but also Types IX, XI, and others) and large molecules of negatively charged, sulfated proteoglycans, as depicted in FIGS. 1 and 1A. The term matrix as used herein refers to a composition which provides structural support for, and which facilitates respiration and movement of nutrients and water to and from, an intervertebral disc. The collagenous components of the nucleus pulposus extracellular matrix comprise a scaffold that provides for normal cell (i.e., chondrocyte) attachment and cell proliferation. The negatively charged proteoglycan component of the nucleus pulposus extracellular matrix attracts water to form a hydrated gel, which envelops the collagen fibrils and chondrocyte cells. In the normal healthy nucleus pulposus, water comprises between 80-90% of the total weight.
The nucleus pulposus thus plays a central role in maintaining normal disc hydrodynamic function. The large molecular weight proteoglycans are contained within the nucleus pulposus by the annulus fibrosus and by the vertebral end plates, and they attract water into the nucleus through sieve-like pores in the end plates. The resulting osmotic pressure within each disc tends to expand it axially (i.e., vertically), driving the adjacent vertebrae further apart. On the other hand, mechanical movements resulting in axial compression, flexion, and rotation of the vertebrae exert forces on the intervertebral discs, which tends to drive water out of the nucleus pulposus. Water movements into and out of an intervertebral disc under the combined influence of osmotic gradients and mechanical forces constitute hydrodynamic functions important for maintaining disc health.
Movement of solutes in the water passing between discs and vertebrae during normal hydrodynamic function facilitates chondrocyte respiration and nutrition within the discs. This function is critical to chondrocyte survival since nucleus pulposus tissues of intervertebral discs are avascular (the largest such avascular structures in the human body). Maintaining sufficient water content in the nucleus pulposus is also important for absorbing high mechanical (shock) loads, for resisting herniation of nucleus pulposus matter under such loads, and for hydrating the annulus fibrosus to maintain the flexibility and strength needed for spine stability.
Normal hydrodynamic functions are compromised in degenerative disc disease (DDD). DDD involves deterioration in the structure and function of one or more interverebral discs and is commonly associated with aging and spinal trauma. Although the etiology of DDD is not well understood, one consistent alteration seen in degenerative discs is an overall decrease in proteoglycan content within the nucleus pulposus and the annulus fibrosus. The loss in proteoglycan content results in a concomitant loss of disc water content. Reduced hydration of disc structures may weaken the annulus fibrosus, predisposing the disc to herniation. Herniation frequently results in extruded nucleus pulposus material impinging on the spinal cord or nerves, causing pain, weakness, and in some cases permanent disability.
Because adequate disc hydration is important for stability and normal mobility of the spine, effective treatment of DDD would ideally restore the disc""s natural self-sustaining hydrodynamic function. Such disc regeneration therapy may require substantial restoration of cellular proteoglycan synthesis within the disc to maintain the hydrated extracellular matrix in the nucleus pulposus. Improved hydrodynamic function in such a regenerated disc may result in restoration and reestablishment of intervertebral disc height. It may also provide for improved hydration of the annulus fibrosus, making subsequent herniation less likely.
Prior art approaches to intervertebral disc problems fail to restore normal self-sustaining hydrodynamic function, and thus may not restore normal spinal stability and/or mobility under high loads. One approach to reforming intervertebral discs using a combination of intervertebral disc cells and a bioactive, biodegradable substrate is described in U.S. Pat. No. 5,964,807 to Gan et al., incorporated herein by reference. The biodegradable substrate disclosed in Gan et al., including bioactive glass, polymer foam, and polymer foam coated with sol gel bioactive material, is intended to enhance cell function, cell growth and cell differentiation. The bioactive glass contains oxides of silicon, sodium, calcium and phosphorus. The polymer foam is described as biocompatible and includes polyglycolide (PGA), poly (D,L-lactide) (D,L-PLA), poly(L-lactide) (L-PLA), poly(D,L-lactide-co-glycolide) (D,L-PLGA), poly(L-lactide-co-glycolide) (L-PLGA), polycaprolactone (PCL), polydioxanone, polyesteramides, copolyoxalates, and polycarbonates. Gan et al. describes application of this approach to intervertebral disc reformation in mature New Zealand rabbits, concluding with ingrowth of cells and concurrent degradation of implanted material with little or no inflammation. However, degradation of portions of the implanted material, such as acidic breakdown of PLAs, PGAs and PLGAs, may adversely affect cell growth, cell function and/or cell differentiation.
A somewhat analogous disclosure relating to tissues for grafting describes matrix particulates comprising growth factors that may be seeded with cells; see U.S. Pat. No. 5,800,537 to Bell, incorporated herein by reference. The matrix and cells are applied to scaffolds, which include biodegradable polymers, microparticulates, and collagen which has been cross-linked by exposure to ultraviolet radiation and formed to produce solids of foam, thread, fabric or film. The matrix particulates are derived from tissue from which cells and cell remnants have been removed without removing factors necessary for cell growth, morphogenesis and differentiation. Bell specifically avoids the use of reagents like high salt, or deliysidation reagents such as butanol/ether or detergents. Such reagents are unfavorably characterized as being responsible for removing from the source tissue factors essential for stimulating repair and remodeling processes. Alternative approaches, in which such factors are obtained from other sources rather than being retained in the tissue, are not addressed.
Still another disclosure related to regeneration of cartilage is found in U.S. Pat. No. 5,837,235 to Mueller et al., incorporated herein by reference. Mueller et al. describes committing small particles of autologous omentum or other fatty tissue for use as a carrier, and adding to the carrier growth factors such as Transforming Growth Factor Beta and Bone Morphogenic Protein. Mueller et al. does not teach cross-linking tissues to create a cross-linked matrix.
The Gan et al. patent above is representative of past attempts to restore or regenerate substantially natural hydrodynamic disc function to intervertebral discs, but such techniques have not been proven in clinical trials. Similarly, the approaches of Bell and Mueller et al. have not been widely adapted for disc regeneration, and better approaches are still needed because low back pain sufficient to prevent the patient from working is said to affect 60% to 85% of all people at some time in their life. In the absence of safer and more efficacious treatment, an estimated 700,000 discectomies and 550,000 spinal fusions are performed worldwide each year to treat these conditions. Several prosthetic devices and compositions employing synthetic components have also been proposed for replacement of degenerated discs or portions thereof. See, for example, U.S. Pat. Nos. 4,772,287, 4,904,260, 5,047,055, 5,171,280, 5,171,281, 5,192,326, 5,458,643, 5,514,180, 5,534,028, 5,645,597, 5,674,295, 5,800,549, 5,824,093, 5,922,028, 5,976,186, and 6,022,376.
A portion of the disc prostheses referenced above comprise hydrogels which are intended to facilitate hydrodynamic function similar in some respects to that of healthy natural discs. See, for example, U.S. Pat. No. 6,022,376 (Assell et al.). These prosthetic hydrogels, however, are not renewed through cellular activity within the discs. Thus, any improvement in disc hydrodynamic function would not be self-sustaining and would decline over time with degradation of the prosthetic hydrogel. Healthy intervertebral discs, in contrast, retain their ability to hydrodynamically cushion axial compressive forces in the spine over extended periods because living cells within the discs renew the natural hydrogel (i.e., extracellular matrix) component.
Restoration of a clinically useful degree of normal hydrodynamic function in degenerated intervertebral discs is an object of the present invention, and the methods and compositions described herein have been shown to induce and/or enhance such regeneration.
The present invention comprises methods and compositions for intervertebral disc regeneration. In preferred embodiments, the compositions comprise a three-dimensional fluid matrix of digestion-resistant, cross-linked nucleus pulposus tissue from a donor vertebrate. The donor may be the patient or another animal of the same or different species. Cross-linking of donor nucleus pulposus tissue for the present invention is preferably achieved through use of one or more photooxidative catalysts which selectively absorb visible light. See U.S. Pat. Nos. 5,147,514, 5,332,475, 5,817,153, and 5,854,397, all incorporated herein by reference. Other cross-linking approaches may be used without departing from the scope of the invention, however.
Prior to cross-linking the tissues, chondrocytes of the donor vertebrate are preferably destroyed, fragmented, and/or removed (i.e., decellularized). A preferred decellularization approach involves soaking the tissue in a solution having high concentrations of salt (preferably NaCl) and sugar (preferably sucrose). Such high-salt, high-sugar solutions are referred to as HSHS solutions. Other decellularization approaches may be used, however. After the tissues are decellularized and cross-linked, the resulting fluid matrix may be lyophilized for sterilization and storage, and then rehydrated prior to use. FIG. 2 illustrates a process for producing a preferred embodiment of the fluid matrix of the present invention.
The fluid matrix of the present invention is biocompatible, substantially non-immunogenic, and resistant to degradation in vivo. As such, it is capable of providing important internal structural support for an intervertebral disc undergoing regeneration during a period of accelerated proteoglycan synthesis. The cross-linked matrix may be delivered to the intervertebral disc space by injection through a syringe (as depicted in FIG. 2), via a catheter, or other methods known in the art.
The three-dimensional fluid matrix of the present invention may be used alone or in combination with growth factors and/or living cells to facilitate regeneration of the structures of a degenerated disc. In patients having sufficient viable endogenous disc cells (chondrocytes) and cell growth factors, the three-dimensional cross-linked matrix alone may substantially contribute to the regeneration of hydrodynamic function in an intervertebral disc in vivo by providing improved mechanical stability of the disc and a more favorable environment for cellular growth and/or metabolism. Conversely, in another embodiment of the invention, a combination of the three-dimensional matrix and one or more purified, preferably bone-derived, cell growth factors may also be used to treat DDD in discs containing viable chondrocytes in a depleted proteoglycan hydrogel matrix. In this case, the cross-linked collagen provides an expanded remodelable three-dimensional matrix for the existing (native) chondrocytes within a disc, while the cell growth factors induce accelerated proteoglycan production to restore the hydrogel matrix of the patient. The combination of the three-dimensional matrix and one or more purified cell growth factors is referred to as a cell growth medium. The present invention may also comprise an injectable cell growth medium. Individual purified cell growth factors may be obtained by recombinant techniques known to those skilled in the art, but a preferred plurality of bone-derived purified cell growth factors for the present invention is disclosed in U.S. Pat. Nos. 5,290,763, 5,371,191 and 5,563,124, all incorporated herein by reference. Bone-derived cell growth factors produced according to these patents are hereinafter referred to as xe2x80x9cBP.xe2x80x9d
Disc regeneration occurs as the cross-linked collagen and proteoglycan matrix supports living cells (which may include exogenous cells as well as native disc or other autologous cells) having inherent capability to synthesize Type II collagen fibrils and proteoglycans in vivo, among other extracellular matrix molecules. Where the patient""s native disc cells have been removed or are otherwise insufficient to cause such proliferation, living cells may be added to the three-dimensional matrix of cross-linked nucleus pulposus material to further promote disc regeneration. Accordingly, in another embodiment, the present invention comprises a three-dimensional matrix of cross-linked nucleus pulposus tissue to which exogenous and/or autologous living cells have been added. The injectable combination of three-dimensional matrix material and exogenous and/or autologous living cells is termed herein an injectable cell matrix. Suitable cells for such an injectable cell matrix may be obtained, for example, from the nucleus pulposus of a mammalian vertebral disc, from cartilage, from fatty tissue, from muscle tissue, from bone marrow, or from bone material (i.e., mesenchymal stem cells), but are not limited to these tissue types. These cells are preferably cultured in vitro to confirm their viability and, optionally, to increase the cells"" proliferation and synthesis responses using cell growth factors.
Growth factors may optionally be added to cell cultures to stimulate cellular development and elaboration of Type II collagen fibrils and proteoglycans suitable for maintaining an effective disc hydrogel matrix in vivo. An injectable fluid combining purified cell growth factors and a plurality of living cells is termed an injectable cell suspension, and is useful in treating DDD. While an injectable cell matrix alone (i.e., without growth factors) may substantially regenerate hydrodynamic function in an intervertebral disc in vivo if sufficient native cell growth factors are present in the disc, purified (exogenous) cell growth factors may be added to an injectable cell matrix of the present invention to form yet another embodiment of the present invention.